Detection and Visualization

Following development, chromatograms are removed from the chamber and are air- or oven-dried to remove the mobile phase, zones are detected by various means. Colored substances may be viewed in daylight without any treatment. Detection of colorless substances is simplest if compounds show self-absorption in the shortwave ultraviolet (UV) region (254 nm) or if they can be excited to produce fluorescence by short-wave and/or by long-wave (365 nm) UV radiation. Otherwise, detection can be achieved by means of chromogenic reagents (producing colored zones), fluorogenic reagents (producing fluorescent zones), or by biological enzymatic methods.

Enzymatic reactions can be monitored on the plate, and the end products can be detected. Biological test procedures are used in the specific detection of biologically active compounds. Thus, detection of hemolyz-ing compounds such as saponins is achieved by casting a blood-gelatin suspension on the layer and observing hemolytic zones that are transparent and nearly colorless on the turbid red gelatin layer background. Another means of detection is the use of Geiger or flow counters or other specialized means to locate radioactive solutes.

Detection reagents may be impregnated into the layer prior to sample application and development. Chromogenic reagents are of two types: (1) general reagents that react with a wide variety of different compound types and can totally characterize an unknown sample, and (2) specific reagents that indicate the presence of a particular compound or functional group. The universal detection reagent iodine can be used as a 1% alcoholic solution spray, but more frequently, the plate is simply placed in a closed container containing a few iodine crystals. The iodine vapor forms weak charge-transfer complexes with most organic compounds which show up as brown spots on a pale yellow background within a few minutes. Sensitivities in the 0.1-0.5-^g range are often obtained with iodine.

Charring reagents (H2SO4) are suitable for glass-backed layers with inorganic (e.g., gypsum) binders only. Many charring reagents produce colored zones when heating is carried out at relatively low temperature; they form black zones at higher temperatures.

Spraying of a chromatogram with a 5% solution of phosphomolybdic acid followed by a brief heating at 110°C gives dark blue spots against a yellow background with a large variety of organic compounds.

A solution of Rhodamine B produces violet spots on a pink background. Antimony trichloride or pentachloride solution in carbon tetrachloride produce spots of different characteristic colors with many organic compounds.

Over 300 spray reagents are known to react more or less specifically with different functional groups to reveal natural products and organic or biochemicals as colored or fluorescent zones. Table IV contains a selection of specific detection reagents. Methods for the quantitation of thin-layer chromatograms can be divided into two categories. In the first, solutes are assayed directly on the layer, either by visual comparison, area measurement or densitometry. In the second, solutes are eluted from the sorbent before being examined further.

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